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D E PA R T E M E N T B I O Z E N T R U M
INTRODUCTION TO FLOW CYTOMETRY
Janine Zankl FACS Core Facility 17. October 2012
Overview or The Many Parts of Flow
• What is Flow Cytometry? • Fluidics • Light Scattering • Fluorescence • Data presentation • Interpreting the data • Applications • Sorting
Flow Basics
Motivation and Experiments
Data Analysis
Cellular Parameters
Lymphocytes Basophils
Monocytes
Eosinophils
Neutrophils
Granulocytes
Cellular Parameters
What Is Flow Cytometry?
• Flow ~ cells in motion • Cyto ~ cell • Metry ~ measure • Measuring properties of cells while in a fluid stream
History and uses of Flow Cytometry
It can be used for… Immunophenotyping DNA cell cycle/tumor ploidy Membrane potential Ion flux Cell viability Intracellular protein staining pH changes Cell tracking and proliferation Sorting Redox state Chromatin structure Total protein Lipids Surface charge Membrane fusion/runover Enzyme activity Oxidative metabolism Sulfhydryl groups/glutathione DNA synthesis DNA degradation Gene expression
0100020003000400050006000700080009000
10000
Publications citing 'flow cytometry'
The use of flow in research has boomed since the mid-1980s
Flow Cytometer
What Happens in a Flow Cytometer?
that is collected, filtered and converted
Cells in suspension
flow in single-file
through an illuminated
volume where they scatter light
and emit fluorescence
to digital values that are stored on a computer
http://flow.csc.mrc.ac.uk/?page_id=5
2. Optics
3. Electronics
1. Fluidics
Fluidics – Hydrodynamic Focusing
• When conditions are right, the sample fluid flows in a central core that does not mix with the sheath fluid
• The introduction of a large volume into a small volume in such a way that it becomes “focused” along an axis is called Hydrodynamic Focusing
Sheath fluid
Injector Tip
sample
Fluorescence Signals
Adapted from Purdue University Cytometry Laboratories
Light Scattering
Forward Scatter FSC small angle scattering (0.5-5°) (roughly) size and shape (blue)
FSC Intensity
coherent light source (488nm, blue)
Light Scattering
Forward Scatter FSC small angle scattering (0.5-5°) roughly size and shape (blue)
FSC Intensity
coherent light source (488nm, blue)
Light Scattering
coherent light source (488nm, blue)
Side Scatter SSC large angle scattering (15-150°), darkfield, complexity and granularity (blue)
SSC Intensity
Light Scattering
coherent light source (488nm, blue)
Side Scatter SSC large angle scattering (15-150°), darkfield, complexity and granularity (blue)
SSC Intensity
Histogram
Forward Scatter FSC
0 10 100 1000 10000
Coun
t
smaller bigger
Dot Plot
Side
Sca
tter
SSC
Forward Scatter FSC
Bigger
Mor
e G
ranu
lar
Live Cells
Bigger Cells or Aggregates
Dead Cells
Apoptotic Cells
Light Scattering in Whole Blood
Colours
• Antibodies • Fluorescent dyes • Fluorescent proteins
NEW HAT? NO FACS
ANTIBODY!
Fluorescence
absorbed excitation
light
emitted fluorescence
light
Fluorescence
coherent light source (488nm)
green and blue fluorescence
Fluorescence emission lower energy than source light (green)
Fluorescence Detetor
Filter Properties
Long-pass filters transmit wavelengths above a cut-on wavelength
Band-pass filters transmit wavelengths in a narrow range around a specified wavelength
550LP
Emission light > 550nm
Emission light
510/20BP
500-520nm
BP LP
Fluorescence
coherent light source (488nm)
green fluorescence intensity
Fluorescence emission lower energy than source light (green)
Fluorescence Detetor
Bandpassfilter
More Fluorescent Parameters…
Dichroic Mirror
At an other angle the filter can be used as a dichroic filter or dichroic mirror
Transmitted Light
Light Source
Filter placed at 45o
Reflected light
Detector E Detector B
Channel Layout of a laser-based Flow Cytometer
Illumination Volume
PI, PerCP
FITC, Alexa 488, GFP
PE
Flow cell
SSC
FSC
• Photon-distribution to detectors according to energy-levels (wavelengths)
• Optical elements provide separation of channels and wavelength selection
Compensation
• Fluorochromes typically fluoresce over a large part of the spectrum • Overlap between the colors emitted by different fluorochromes • Mathematical compensation is used to reduce overlapping results
overlapping emmission spectra wrong positive signal
Compensation
• Fluorochromes typically fluoresce over a large part of the spectrum • Overlap between the colors emitted by different fluorochromes • Mathematical compensation is used to reduce overlapping results
green fluorescence
yello
w f
luor
esce
nce
uncompensated
correctly compensated
green fluorescence
yello
w f
luor
esce
nce
Microscopy vs Flow Cytometry
Microscopy • Localization of antigen is possible • Poor enumeration of cell subtypes • Limiting number of simultaneous
measurements
Flow Cytometry • Can not tell you where antigen is • Fast • Subpopulation analysis • Multiparameter analysis
N-Dimensional Plots
Gating
• Is used to isolate a subset of cells on a plot
• Allows the ability to look at parameters specific to only that subset
green
green red both
red
apoptotic
dead
Mixed T cell population
3% double pos. 60% double pos.
Important Points on Analysis
• Visualization
• What kind of data are you looking for?
• How much fluorescence? • What percent are positive? • How much more positive is x
than y? • What is the ratio between
green and red fluorescence?
• What kind of statistics are available
• MFI (geometric or arithmetic) • %-ages • CV • …
Contour Plot Pseudo-colour Density
Greyscale Density Dot Plot
www.treestar.com
Applications of Flow Cytometry
Cell function
DNA and RNA analysis
Cell death Sorting and cell isolation
Immunopheno-typing main
applications
Application: Immunophenotyping
• Detection of cell surface molecules as example cluster of differentiation
Lymphocyte T or B????
only cells from R1
SSC
FSC
www.med4you.at
T-cell marker
B-ce
ll m
arke
r
Application: Clinical phenotyping
Blood from Patient
Normal blood sample
www.med4you.at
Application: Clinical phenotyping
www.med4you.at
Blood from Patient
Normal blood sample
Application: Cell function
• Intracellular staining of cytokines, cytoskeleton, enzymes, transcription factors, signaling molecules Fixation
Permeabilization
Application: DNA Analysis
• DNA content of individual cells gives information about their ploidy • Suitable dyes: PI, 7-AAD, DAPI • Combination with light scatter or immunofluorescence
Application: Cell Death
Measurements of cell death: • Expression of proteins involved
in apoptosis • Activation of caspases • Changes in the mitochondrial
membrane potential • Changes in the plasma
membrane • Cell shrinkage • Chromatin changes • DNA degradation
Normal HL60
apoptotic HL60
Time in hours Time in hours
Application: Cell Expression
FSC SSC
SSC
DAPI
dead cells
Coun
t Ye
llow
Flu
ores
cenc
e In
tens
ity
Green Fluorescence Intensity
Principle of Sorting
• Sorting (separating) cells based on properties measured in flow is also called Fluorescence-Activated Cell Sorting FACS
Application: Sorting
green fluorescence
red
fluor
esce
nce
dead cells viable cells
analysis sort
green fluorescence
yello
w fl
uore
scen
ce culture
sort
Advantages and Disadvantages
₊ Distinct cell populations defined by size and granularity ₊ Dead cells may be gated out for analysis ₊ Detection of weakly expressed surface antigens ₊ Multicolor analysis may be performed
₋ Sterility issues: antibiotics sufficient to control microbial contamination ₋ Viability of fragile cells : larger nozzle, 4°C, appropriate buffer
What are the services in our facility
Analysis • BD Canto II • Introduction 1h • Booking / usage by yourself • Advice if requested
Sorting • BD AriaIII • Appointment for experiment
discussion • Check cells for fluorescence • Prepare the cells and let them sort
Summary
isac-net.org www.cyto.purdue.edu
flowbook-wiki.denovosoftware.com www.bdbiosciences.com/research/multicolor/spectrum_viewer/index.jsp
www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence-SpectraViewer.html
Links:
janine.zankl@unibas.ch
Electronics – Voltage Pulse
Laser Beam
Transmitted Light
Detector
Volta
ge
Time
Measurements of the Electronic Signal
Volta
ge
Time
Area A
High H
Width W
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